This invention relates to methods and apparatus for performing capillary electrophoresis. More specifically, this invention is directed towards methods and apparatus useful for reducing peak broadening caused during the establishment of a run field used to conduct the capillary electrophoresis process.
Electrophoretic separations of biopolymers and small molecules are critically important in modern biology and biotechnology, playing a central role in such techniques as DNA sequencing, protein molecular weight determination, genetic mapping, and the like. A particularly preferred electrophoresis format is capillary electrophoresis (CE), where the electrophoresis is performed in a capillary channel having a small internal diameter, e.g., between 5 and 100 xcexcm. In many applications, capillary electrophoresis results in enhanced separation performance over traditional slab-based formats because the superior ability of the narrow-bore capillary to dissipate Joule heat allows for the use of high electrical fields thereby resulting in fast separations in which the effect of analyte diffusion is reduced. In addition, capillary electrophoresis is well adapted to automation because of the ability to automate the steps of sample loading, analyte detection, and replenishment of the separation medium.
Certain commercially important applications of capillary electrophoresis require exquisite separation efficiency. For example, in DNA sequencing separations, plate counts of 20 million plates per meter may be required. In order to achieve this kind of performance, everything possible must be done to reduce instrumental effects that can lead to peak broadening and therefore lower separation efficiencies, e.g., peak broadening caused by the radial temperature profile within the lumen of the capillary, the sample injection volume, solute-wall interactions, siphoning, finite detection volume, and the like (e.g., Capillary Electrophoresis Theory and Practice, Chap. 1, Grossman and Colburn, eds., Academic Press (1992)). In addition, because of the high throughput requirements of large-scale DNA sequencing operations, any measures taken to increase the separation performance of the electrophoretic analysis preferably will not substantially reduce the speed, and therefore the throughput, of the process.
Therefore, any further understanding of the mechanisms underlying peak broadening and techniques for reducing the impact of such mechanisms on the performance of CE separations without sacrificing the speed of analysis would be an important contribution to the field of capillary electrophoresis and related applications.
The present invention is directed towards the discovery of methods and apparatus useful for increasing the separation performance of capillary electrophoresis separations performed in a fluid separation medium by controlling the rate of increase of the electric field strength and/or the temperature of the separation medium during an initial-electric-field-ramp stage of the capillary electrophoresis process.
In one aspect, the invention comprises a capillary electrophoresis method wherein a run field is established during an initial electric field ramp in a controlled manner according to a pre-defined ramp rate. In a preferred embodiment of this aspect of the invention, the run field is established at a ramp rate no greater than about 5 V/cm-s. In another preferred embodiment of this aspect of the invention, the run field is established over a period of at least about ten seconds. In yet another preferred embodiment of this aspect of the invention, the run field is established at a ramp rate which results in a reduction in the amount of peak broadening associated with the establishment of the run field by at least about 10%. In another preferred embodiment of this aspect of the invention, the run field is established at a ramp rate which results in an increase in a length of read of at least about 20 nucleotides over that achieved when the run field is not established in a controlled manner.
In another aspect, the invention comprises a method for producing a desired reduction in an amount of peak broadening caused during establishment of a run field comprising, for each of a plurality of electrophoretic runs, establishing the run field at each of a plurality of different ramp rates, at least some of which ramp rates are not greater than about 5 V/cm-s; analyzing a degree of peak broadening observed for each run; and selecting a ramp rate which is no greater than that which produced a desired reduction in peak broadening.
In yet another aspect, the present invention comprises a capillary electrophoresis method in which analyte species are separated by differential electrophoretic migration through a fluid separation medium located within a capillary under the influence of a run field, an improvement for reducing the peak broadening associated with the establishment of the run field comprising reducing a temperature of an environment surrounding the capillary during an initial electric field ramp. In one preferred embodiment, the temperature of the environment surrounding the capillary is reduced by an amount sufficient to maintain an average temperature of the separation medium during such initial electric field ramp to within about 0.4xc2x0 C. of the average temperature of the separation medium prior to initiating the initial electric field ramp. In another preferred embodiment, the temperature of the environment surrounding the capillary is reduced by an amount sufficient to maintain an average temperature of the separation medium during such initial electric field ramp substantially constant with respect to an average temperature of the separation medium prior to initiating the initial electric field ramp to an extent sufficient to result in a displacement of the fluid separation medium at an inlet end of the capillary during the initial electric field ramp of less than about 600 xcexcm. In yet another preferred embodiment, the temperature of the environment surrounding the capillary is reduced by an amount sufficient to maintain an average temperature of the separation medium during such initial electric field ramp substantially constant with respect to an average temperature of the separation medium prior to initiating the initial electric field ramp to an extent sufficient to increase a length of read by at least about 20 nucleotides.
These and other aspects, embodiments and features of the present invention will become better understood with reference to the following description, drawings, and appended claims.